This invention relates to cardiovascular disease.
Cardiovascular disease, the principal cause of death in the developed world, affects men far more frequently than premenopausal women of the same age. Because the incidence of cardiovascular disease in postmenopausal women gradually approaches that in age-matched men (McGill et al., Atheroscler. Rev. 4, 157-242 (1979)), estrogen or progesterone appears to have a protective effect in premenopausal women. A large, 10-year prospective study supports such a cardioprotective effect of estrogen by showing that postmenopausal estrogen-replacement therapy reduces both the incidence of coronary heart disease and mortality from cardiovascular disease (Stampfer, M. J. et al., N. Engl. J. Med. 325, 756-762 (1991)).
As for the effects of progesterone on cardiovascular disease, however, there has been little information until the recent report by Grodstein et al. (N. Engl. J. Med. 335, 453-461 (1996)) showing that the risk of coronary heart disease is significantly lower in women who take estrogen and progesterone together rather than estrogen alone. Estrogen administration inhibits the development of experimentally induced atherosclerosis in rodent models (Stamler et al., Circ. Res. 1, 94-98 (1953); Weigensberg et al., Atherosclerosis 52, 253-265 (1984); and Sullivan et al., J. Clin. Invest. 96, 2482-2488 (1995)). Also, in castrated baboons receiving estradiol and progesterone together, there are fewer vascular lesions than in those receiving estradiol alone (Kushwaha et al., Arterioscler. Thromb. 11, 23-31 (1991)). However, there is little evidence of an independent effect of progesterone in animal studies or in cell culture.
Current methods of treating severe cardiovascular conditions, such as coronary stenosis and occlusion caused by atherosclerosis, include invasive cardiovascular surgical procedures [e.g., percutaneous translumenal coronary angioplasty (xe2x80x9cPTCAxe2x80x9d) and aorta-coronary bypass surgery (xe2x80x9cACBSxe2x80x9d)]. However, the cellular proliferative response and associated intimal hyperplasia that may follow the damage to blood vessels caused by these procedures leads to complications which cannot be effectively controlled by presently available drugs, and can therefore be more detrimental than the original condition. The development of these complications, termed restenosis (in the case of PTCA) or stenosis (in the case of ACBS), has similarities to the development of atherosclerosis.
The invention is based on Applicants"" discovery that (1) progesterone has a direct and specific inhibitory effect on arterial smooth muscle cell proliferation; (2) this inhibition occurs at a progesterone concentration that overlaps the physiological range of progesterone concentrations in the plasma of premenopausal women; (3) arterial smooth muscle cells contain abundant progesterone receptors (xe2x80x9cPgRxe2x80x9d) that mediate this effect; and (4) in the presence of progesterone, mRNA levels of cyclins A and E decline in rat arterial smooth muscle cells (xe2x80x9cRASMCxe2x80x9d), suggesting that progesterone interrupts the cell cycle at the G1/S transition. To Applicants"" knowledge, this is the first demonstration that progesterone exerts a direct inhibitory effect on vascular smooth muscle cell proliferation.
Applicants"" discovery provides a biochemical explanation for the recent epidemiological observation that the relative risk of major coronary heart disease was 0.39 in postmenopausal women who took estrogen with progesterone, as compared with a risk of 0.60 in those who took estrogen alone (where the risk among women who took no hormones was set at 1.0) (Grodstein et al, supra).
Accordingly, the present invention features a method of treatment. In this method, one first identifies a mammal that is suspected of having, or at a risk of having, a condition characterized by vascular smooth muscle cell (xe2x80x9cVSMCxe2x80x9d) proliferation and that is not being treated with estrogen or an estrogen agonist. Such a mammal can, for example, be a mouse, rat, dog, cat, cow, pig, horse, goat, sheep, rabbit, guinea pig, hamster or primate, such as a an adult human (e.g., a man, a premenopausal woman, or a postmenopausal woman who is not presently on an estrogen replacement therapy). A mammal is at a risk of having the condition when it, for example, is at an age of being susceptible to the condition, or is genetically predisposed to or has a family history of the condition, or is about to undergo or has recently undergone vascular surgery. A condition marked by VSMC proliferation is typically a cardiovascular or coronary heart disease such as transplant arteriosclerosis, atherosclerosis, angioplasty restenosis, or cardiac vein bypass stenosis.
After such a mammal is identified, one can then administer to the mammal progesterone or a progesterone agonist in an amount effective to decrease VSMC proliferation in the mammal. A progesterone agonist is a compound that mimics, to various degrees, the effects of progesterone. This compound can be, for instance, an organic compound, a peptide, or a nucleic acid.
An amount of progesterone or an agonist thereof is effective when its administration results in inhibition of VSMC proliferation in a blood vessel in the mammal.
Also embraced by the invention are methods of identifying compounds potentially useful for inhibiting VSMC proliferation. In these methods, a test compound is contacted with a progesterone receptor (PgR). There are at least two isoforms of PgRs: PgR A and PgR B, the molecular weights of which are approximately 94 and 114 kD, respectively. The test compound""s binding to the PgR is an indication that the compound is potentially useful for inhibiting vascular smooth muscle cell proliferation. The PgR can be in a purified protein preparation, in a cellular extract, or inside cells [e.g., VSMCs or cells (e.g., mammalian, yeast, insect, or bacterial cells) that have been transfected with a DNA construct directing expression of the PgR].
Alternatively, the potential usefulness of a test compound is indicated by its ability to activate the PgR in a cell or in a cell extract. Activation of the PgR is detected, for example, by the phosphorylation status of the PgR, or by enhanced transcription of a gene that is linked to a cis progesterone-responsive DNA element (e.g., the hormone response element of the mouse mammary tumor virus). This gene can be an endogenous gene such as cyclin A or E, whose transcription is downregulated by activated progesterone receptors (see Example, infra). Alternatively, the gene is an exogenously introduced reporter gene whose expression can be readily determined by a biochemical assay (e.g., a gene that encodes a firefly luciferase, xcex2-galactosidase, or alkaline phosphatase) or by drug resistance of the cell harboring the gene (e.g., a gene that encodes chloramphenicol acetyltransferase, neomycin phosphotransferase, or guanine xanthine phosphoribosyltransferase).
The invention also provides methods of determining whether compounds known or suspected to bind a PgR, such as known progesterone agonists (e.g., norethisterone, or norgestrel), are potentially useful for inhibiting VSMC proliferation. In these methods, mammalian cells (e.g., mammalian VSMCs) containing a PgR are incubated with the compounds and relative proliferation of the cells is determined (e.g., by the amount of radioactive nucleotides incorporated into replicating genomic DNA, or by the number of cells generated by cell division). The potential usefulness of PgR-binding compounds can also be determined by measuring their ability to decrease cyclins A and/or E activity or expression level (e.g., amounts of proteins or mRNA transcripts) in mammalian cells (e.g., mammalian VSMCs) containing a PgR.
Cells used to practice the invention can be freshly isolated from tissues or cultured from isolated tissue cells. The cells can also be those that have been immortalized in vivo (i.e., cancer cells) or in vitro. A PgR used in the invention can be either naturally occurring (i.e., encoded by an endogenous gene) or recombinant, and need not be full length, so long as it contains the domains essential for its functions.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.